scholarly journals Female Site-Specific Transposase-Induced Recombination: A High-Efficiency Method for Fine Mapping Mutations on the X Chromosome in Drosophila

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 591-597 ◽  
Author(s):  
Jeffrey M Marcus
Keyword(s):  
X Chromosome ◽  
Meiotic Recombination ◽  
High Efficiency ◽  
P Element ◽  
Male Recombination ◽  
Site Specific ◽  
Site Specific Recombination ◽  
P Elements ◽  
Male Germline ◽  
Meiotic Mapping

Abstract P-element transposons in the Drosophila germline mobilize only in the presence of the appropriate transposase enzyme. Sometimes, instead of mobilizing completely, P elements will undergo site-specific recombination with the homologous chromosome. Site-specific recombination is the basis for male recombination mapping, since the male germline does not normally undergo recombination. Site-specific recombination also takes place in females, but this has been difficult to study because of the obscuring effects of meiotic recombination. Using map functions, I demonstrate that it is possible to employ female site-specific transposase-induced recombination (FaSSTIR) to map loci on the X chromosome and predict that FaSSTIR mapping should be more efficient than meiotic mapping over short genetic intervals. Both FaSSTIR mapping and meiotic mapping were used to fine map the crossveinless locus on the X chromosome. Both techniques identified the same 10-kb interval as the probable location of the crossveinless mutation. Over short intervals (< ∼7.6 cM), FaSSTIR produces more informative recombination events than does meiotic recombination. Over longer intervals, FaSSTIR is not always more efficient than meiotic mapping, but it produces the correct gene order. FaSSTIR matches the expectations suggested by the map functions and promises to be a useful technique, particularly for mapping X-linked loci.

Mapping of Drosophila Mutations Using Site-Specific Male Recombination

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 157-163
Author(s):  
Bin Chen ◽  
Tehyen Chu ◽  
Emily Harms ◽  
J Peter Gergen ◽  
Sidney Strickland
Keyword(s):  
Chromosomal Region ◽  
P Element ◽  
Male Recombination ◽  
Site Specific ◽  
Site Specific Recombination ◽  
P Elements ◽  
Element Insertion ◽  
Insertion Sites ◽  
General Method

Abstract Although recombination does not usually occur in the male Drosophila germline, site-specific recombination can be induced at the ends of P elements. This finding suggested that male recombination could be used to map Drosophila mutations. In this article, we describe the general method and its application to the mapping of two EMS-induced female-sterile mutations, grauzone and cortex. Within two months, the grauzone gene was mapped relative to seven different P-element insertion sites, and cortex was mapped relative to 23 different P-elements. The results allowed us to map grauzone to a region of about 50 kb, and cortex distal to the chromosomal region 33E. These experiments demonstrate that P-element-induced site-specific male recombination is an efficient and general method to map Drosophila autosomal mutations.

scholarly journals Telomeric P elements Associated With Cytotype Regulation of the P Transposon Family in Drosophila melanogaster

Genetics ◽  
2002 ◽  
Vol 162 (4) ◽  
pp. 1641-1654 ◽  
Author(s):  
Jeremy R Stuart ◽  
Kevin J Haley ◽  
Douglas Swedzinski ◽  
Samuel Lockner ◽  
Paul E Kocian ◽  
...  
Keyword(s):  
X Chromosome ◽  
Gonadal Dysgenesis ◽  
Molecular Level ◽  
P Element ◽  
Wild Type ◽  
Maternal Transmission ◽  
P Elements ◽  
Male Germline ◽  
Males And Females ◽  
P Transposon

Abstract P elements inserted at the left end of the Drosophila X chromosome were isolated genetically from wild-type P strains. Stocks carrying these elements were tested for repression of P-strain-induced gonadal dysgenesis in females and for repression of transposase-catalyzed P-element excision in males and females. Both traits were repressed by stocks carrying either complete or incomplete P elements inserted near the telomere of the X chromosome in cytological region 1A, but not by stocks carrying only nontelomeric X-linked P elements. All three of the telomeric P elements that were analyzed at the molecular level were inserted in one of the 1.8-kb telomere-associated sequence (TAS) repeats near the end of the X chromosome. Stocks with these telomeric P elements strongly repressed P-element excision induced in the male germline by a P strain or by the transposase-producing transgenes H(hsp/CP)2, H(hsp/CP)3, a combination of these two transgenes, and P(ry+, Δ2-3)99B. For H(hsp/CP)2 and P(ry+, Δ2-3)99B, the repression was also effective when the flies were subjected to heat-shock treatments. However, these stocks did not repress the somatic transposase activity of P(ry+, Δ2-3)99B. Repression of transposase activity in the germline required maternal transmission of the telomeric P elements themselves. Paternal transmission of these elements, or maternal transmission of the cytoplasm from carriers, both were insufficient to repress transposase activity. Collectively, these findings indicate that the regulatory abilities of telomeric P elements are similar to those of the P cytotype.

scholarly journals A hobo Transgene That Encodes the P-Element Transposase in Drosophila melanogaster: Autoregulation and Cytotype Control of Transposase Activity

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 195-204 ◽  
Author(s):  
Michael J Simmons ◽  
Kevin J Haley ◽  
Craig D Grimes ◽  
John D Raymond ◽  
Jarad B Niemi
Keyword(s):  
Drosophila Melanogaster ◽  
Gonadal Dysgenesis ◽  
P Element ◽  
Hsp70 Gene ◽  
Alternate Splicing ◽  
Male And Female ◽  
P Elements ◽  
Male Germline ◽  
Female Germline ◽  
Transposase Expression

Abstract Drosophila were genetically transformed with a hobo transgene that contains a terminally truncated but otherwise complete P element fused to the promoter from the Drosophila hsp70 gene. Insertions of this H(hsp/CP) transgene on either of the major autosomes produced the P transposase in both the male and female germlines, but not in the soma. Heat-shock treatments significantly increased transposase activity in the female germline; in the male germline, these treatments had little effect. The transposase activity of two insertions of the H(hsp/CP) transgene was not significantly greater than their separate activities, and one insertion of this transgene reduced the transposase activity of P(ry+, Δ2-3)99B, a stable P transgene, in the germline as well as in the soma. These observations suggest that, through alternate splicing, the H(hsp/CP) transgene produces a repressor that feeds back negatively to regulate transposase expression or function in both the somatic and germline tissues. The H(hsp/CP) transgenes are able to induce gonadal dysgenesis when the transposase they encode has P-element targets to attack. However, this ability and the ability to induce P-element excisions are repressed by the P cytotype, a chromosomal/cytoplasmic state that regulates P elements in the germline.

scholarly journals Chromosome interactions in P-M dysgenic male recombination of Drosophila melanogaster

1992 ◽  
Vol 60 (3) ◽  
pp. 165-174
Author(s):  
P. Eggleston ◽  
K. A. Exley
Keyword(s):  
Drosophila Melanogaster ◽  
Recombination Breakpoint ◽  
P Element ◽  
Chromosome 2 ◽  
Male Recombination ◽  
P Elements ◽  
Element Array ◽  
Recombination Breakpoints ◽  
Map Distance

SummaryThe frequency, distribution and structure of P elements on the second and third chromosomes of Texas 1, a wild-type inbred strain of Drosophila melanogaster, were investigated by in situ hybridization. These autosomes were isolated individually and used as P-element donors to study the frequency and distribution of male recombination events generated on recipient chromosomes which were originally devoid of P sequences. The P-element array of chromosome 2 was shown to generate higher male recombination frequencies on chromosome 3 than vice versa, despite having fewer P factors and fewer P elements in general. This is likely to be due to the presence and distribution of specific P-deletion derivatives, which vary in their ability to repress P mobility. The male recombination generated on recipient chromosomes is associated with the insertion of donated P sequences, but only in a small minority of cases could a novel P-element site be detected at, or near, the recombination breakpoint. The majority of such breakpoints appear to be associated either with unsuccessful P insertion, or with the action of P transposase attracted by P elements newly inserted elsewhere on the recipient chromosome. Recent evidence also suggests that a small proportion of the breakpoints may be associated with the action of P transposase alone. Male recombination breakpoints appear to be distributed effectively at random along the recipient autosomes, and their frequency of occurrence was shown to correlate with the physical length of DNA available between markers, as revealed by the polytene map distance.

scholarly journals Local transposition of P elements in Drosophila melanogaster and recombination between duplicated elements using a site-specific recombinase.

Genetics ◽  
1994 ◽  
Vol 137 (2) ◽  
pp. 551-563 ◽  
Author(s):  
K G Golic
Keyword(s):  
Drosophila Melanogaster ◽  
Sister Chromatid ◽  
P Element ◽  
White Gene ◽  
Direct Repeats ◽  
Site Specific ◽  
P Elements ◽  
Dicentric Chromosomes ◽  
A Site ◽  
Different Levels

Abstract The transposase source delta 2-3(99B) was used to mobilize a P element located at sites on chromosomes X, 2 and 3. The transposition event most frequently recovered was a chromosome with two copies of the P element at or near the original site of insertion. These were easily recognized because the P element carried a hypomorphic white gene with a dosage dependent phenotype; flies with two copies of the gene have darker eyes than flies with one copy. The P element also carried direct repeats of the recombination target (FRT) for the FLP site-specific recombinase. The synthesis of FLP in these flies caused excision of the FRT-flanked white gene. Because the two white copies excised independently, patches of eye tissue with different levels of pigmentation were produced. Thus, the presence of two copies of the FRT-flanked white gene could be verified. When the P elements lay in the same orientation, FLP-mediated recombination between the FRTs on separated elements produced deficiencies and duplications of the flanked region. When P elements were inverted, the predominant consequence of FLP-catalyzed recombination between the inverted elements was the formation of dicentric chromosomes and acentric fragments as a result of unequal sister chromatid exchange.

scholarly journals The relationship between P elements and male recombination in Drosophila melanogaster.

Genetics ◽  
1990 ◽  
Vol 124 (2) ◽  
pp. 317-329
Author(s):  
A Duttaroy ◽  
M McCarron ◽  
K Sitaraman ◽  
G Doughty ◽  
A Chovnick
Keyword(s):  
Drosophila Melanogaster ◽  
Hot Spot ◽  
The Other ◽  
P Element ◽  
Male Recombination ◽  
P Elements ◽  
Selective System ◽  
Element Mobilization ◽  
The Relationship ◽  
Initial Binding

Abstract P element dysgenesis associated male recombination in Drosophila was examined with a selective system focused upon 5% of the standard female genetic map divided into eight recombination segments. We found no correspondence between P element mobilization events and recombination in males in the intervals monitored. We defined two adjacent short genetic and molecular regions, one devoid of male recombination and the other acting as a "hot spot" for exchange in the absence of supporting P element insertion and excision activity. These data suggest that, even in the presence of mobilizing P elements, transposase may be active at non-P element sites, and that the genome may harbor sequences ranging from highly responsive to completely unresponsive to transposase action. A viewpoint is presented wherein P elements, with sequences that bind transposase, serve to focus the recombination action of transposase to encompass a region of DNA radiating outward from the initial binding site. We suggest that this region is measured in terms of chromosomal segments rather than limited to P element sequences.

scholarly journals Germ-line and somatic recombination induced by in vitro modified P elements in Drosophila melanogaster.

Genetics ◽  
1990 ◽  
Vol 124 (2) ◽  
pp. 331-337 ◽  
Author(s):  
J A Sved ◽  
W B Eggleston ◽  
W R Engels
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Somatic Cells ◽  
Chromosome Breakage ◽  
P Element ◽  
Male Germ Line ◽  
Male Recombination ◽  
P Elements ◽  
Somatic Recombination

Abstract The P element insertion delta 2-3(99B) has previously been shown to activate incomplete P elements elsewhere in the genome. We show that this element, in conjunction with a second incomplete P element, P[CaSpeR], also induces recombination in the male germ line. The recombination is induced preferentially in the region of the P[CaSpeR] element. Recombinant chromosomes contain the P[CaSpeR] element in more than 50% of cases, and alternative models of transposon replication and preferential chromosome breakage are put forward to explain this finding. As is the case with male recombination induced by P-M dysgenic crosses, recombination appears to be premeiotic in a high proportion of cases. The delta 2-3(99B) element is known to act in somatic cells. Correspondingly, we show that the delta 2-3(99B)-P[CaSpeR] combination elevates the incidence of somatic recombination.

scholarly journals Genetic and molecular analysis of repression in the P–M system of hybrid dysgenesis in Drosophila melanogaster

1991 ◽  
Vol 57 (3) ◽  
pp. 213-226 ◽  
Author(s):  
Ellen M. Heath ◽  
Michael J. Simmons
Keyword(s):  
Drosophila Melanogaster ◽  
Maternal Effects ◽  
X Chromosome ◽  
Gonadal Dysgenesis ◽  
Inbred Lines ◽  
Hybrid Dysgenesis ◽  
P Element ◽  
P Elements ◽  
Two Generations ◽  
Highly Correlated

SummaryTwelve inbred lines derived from an M′ strain of Drosophila melanogaster were used to study the repression of P-element-mediated hybrid dysgenesis. Initial assessments indicated that the lines differed in the ability to repress gonadal dysgenesis, and that this ability was highly correlated with the ability to repress snw hypermutability. Later assessments indicated that most of the lines with low or intermediate repression potential evolved to a state of higher repression potential; however, Southern analyses failed to reveal significant changes in the array of genomic P elements that could account for this evolution. In addition, none of the lines possessed the incomplete P element known as KP, which has been proposed to explain repression in some D. melanogaster strains. One of the lines maintained intermediate repression potential throughout the period of study (52 generations), indicating that the intermediate condition was not intrinsically unstable. Genetic analyses demonstrated that in some of the lines, repression potential was influenced by factors that were inherited maternally through at least two generations; however, these factors were not as influential as those in a classic P cytotype strain. Additional tests with a dysgenesis-inducing X chromosome called T-5 indicated that repression itself was mediated by a combination of maternal effects and paternally inherited factors that were expressed after fertilization. These tests also suggested that in some circumstances, the P transposase, or its message, might be transmitted through the maternal cytoplasm.

Transgene Coplacement and High Efficiency Site-Specific Recombination With the Cre/loxP System in Drosophila

Genetics ◽  
1996 ◽  
Vol 144 (2) ◽  
pp. 715-726 ◽  
Author(s):  
Mark L Siegal ◽  
Daniel L Hartl
Keyword(s):  
Germ Cells ◽  
Maternal Effect ◽  
High Efficiency ◽  
Loxp Site ◽  
Position Effects ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Bacteriophage P1 ◽  
Eye Color ◽  
F2 Generation

Abstract Studies of gene function and regulation in transgenic Drosophila are often compromised by the possibility of genomic position effects on gene expression. We have developed a method, called transgene coplacement, in which any two sequences can be positioned at exactly the same site and orientation in the genome. Transgene coplacement makes use of the bacteriophage P1 system of Cre/loxP site-specific recombination, which we have introduced into Drosophila. In the presence of a cre transgene driven by a dual hsp70-Mosl promoter, a white reporter gene flanked by loxP sites is excised with virtually 100% efficiency both in somatic cells and in germ cells. A strong maternal effect, resulting from Cre recombinase present in the oocyte, is observed as white or mosaic eye color in F1 progeny. Excision in germ cells of the F1 yields a strong grand-maternal effect, observed as a highly skewed ratio of eye-color phenotypes in the F2 generation. The excision reactions of Cre/loxP and the related FLP/FRT system are used to create Drosophila lines in which transgenes are at exactly allelic sites in homologous chromosomes.

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